Melanomas are one the most aggressive tumors with increasing incidence and mortality. Although melanomas are immunogenic tumors that could be potentially eliminated by immune-mediated spontaneous regression, this phenomenon is extremely rare. More frequently, melanomas are not recognized by the immune system due to the absence of tumor-specific antigens (Ags) or development of a state of tumor-specific tolerance. Immunization strategies against melanoma by means of DNA vaccines are promising therapeutic tools based on the induction of tumor-specific CD4+ T helper 1 (Th1) lymphocytes and CD8+ cytotoxic T cells (CTLs). Due to its easy accessibility and intrinsic immune system, the skin is an optimal site for genetic immunization and the gene gun (GO is an excellent approach to induce high expression of transgenic Ags in the skin. Professional Ag processing and presenting cells of the skin [cutaneous dendritic cells (DCs): epidermal Langerhans cells and dermal DCs] can be transfected in situ by GG-immunization, or alternatively, they may internalize transgenic proteins from neighboring skin cells. Therefore, through direct of indirect transfection of skin DCs by the GG, it is possible to engineer in vivo the skin immune system. The presence of appropriate immuno-stimulatory signals during the early stages of DCs activation in peripheral tissues (i.e. skin) "instructs" DCs to fully mature and to induce an effective anti-tumor Th1-biased immune response. However, although the GG induces anti-tumor CD8+ CTLs, it is still controversial whether the GG is able to generate an effective CD4+ Th1-biased immune response (required to maintain efficient cellular anti-tumor immunity) and it rather induces Th2 cells. The hypothesis of this proposal is that the use of the GG to express transgenic melanoma Ags in the skin in the presence of the Th1-driving adjuvants substance P neurokinin 1 receptor agonist (SP-NK1a) and Imiquimod (to mature and to Th1-polarize skin DCs at the site of GG-immunization) will generate fully activated skin DCs with ability to stimulate efficiently tumor Ag-specific T cells. Skin-derived DCs generated under these conditions will bias the differentiation of naive tumor-specific T cells into Th1 cells and promote development of CTLs, generating an effective and long-lasting anti-tumor immune response. To test the hypothesis, we propose the following specific aims: Aim 1: To investigate the function of DCs migrated from skin transfected with GG in the presence of Th1-driving adjuvants Imiquimod and SP-NK1a. Aim 2: To analyze in vivo the role of Th1-driving adjuvants in the outcome of the immune responses induced by GG genetic immunization of skin, and Aim 3: To analyze the capacity of the Th1-driving adjuvants to polarize different subpopulations of skin migratory DCs from human skin transfected with the GG.